Cells continuously synthesize proteins from and degrade them into their component amino acids. The function of this seemingly wasteful process is twofold: first, to eliminate abnormal proteins whose accumulation would be harmful to the cell, and second, to permit the regulation of cellular metabolism by eliminating superfluous enzymes and regulatory proteins. The level of an enzyme depends on its rate of degradation as well as its rate of synthesis. Non-selective protein degradation occurs by a lysosomal mechanism. In eukaryotes a cytosolic ATP-dependent mechanism of protein degradation involves ubiquitin. This mechanism is based on the protein's half life which is partially determined by its N-terminal residue, giving this degradation mechanism the name of N-end rule pathway. The enzymes included in this application are involved in the N-end rule pathway of protein degradation.
Arginyl-tRNA-protein transferase (EC 2.3.2.8) is involved in catalyzing the post-translational conjugation of arginine to the amino termini of acceptor proteins. The function of these enzymes in eukaryotes is apparently to conjugate destabilizing amino acids to the amino termini of short-lived proteins. This reaction is a part of the N-end rule pathway of protein degradation. Arabidopsis thaliana encodes a single form of arginyl-tRNA-protein transferase while two forms, which are differentially spliced, are encoded by mice and humans (Kwon et al. (1999) Mol. Cell. Biol. 19:182-193). A soybean EST encoding a peptide with similarities to cDNAs encoding arginyl-tRNA-protein transferase is found in the NCBI EST database having NCBI General Identifier No.4313355.
The 26S proteosome is the central protease of the ubiquitin-dependent pathway of protein degradation. The 26S proteosome is formed by a barrel-shaped 20S core complex and two polar 19S complexes. The 20S particle contains the protease activity while the 19S complex contains isopeptidase, ATPase, and protein unfolding activities (Koster et al. (1995) Mol. Biol. Rep. 21:11-20). Isopeptidases, also called ubiquitin carboxy-terminal hydrolases, belong to a family containing at least 10 different members. A 100 kDa de-ubiquitinating enzyme isolated from human tissues has been shown to contain "His and Cys domains" which are likely involved in the de-ubiquitinating activity, and an aspartic acid domain of unknown function (Falquet et al. (1995) FEBS Lett. 376:233-237). Because this enzyme cleaves peptide-linked and isopeptide-linked ubiquitin moieties from substrates, it is considered a de-ubiquitinase, instead of a isopeptidase (Falquet et al. (1995) FEBS Lett. 359:73-77). An alignment of de-ubiquitinating enzymes shows that de-ubiquitinase is 99% similar to isopeptidase T. De-ubiquitinase contains a 23 amino acid insertion at position 629 of isopeptidase T which may account for the difference in activity between the two enzymes. Isopeptidase T is a monomeric ubiquitin-binding protein whose activity is inhibited by iodoacetamide and ubiquitin aldehyde (Hadari et al. (1992) J. Biol. Chem. 267:719-727). A corn EST encoding a peptide with similarities to cDNAs encoding isopeptidase T is found in the NCBI database having NCBI General Identifier No. 4688518. Rice ESTs encoding peptides with similarities to cDNAs encoding isopeptidase T are found in the NCBI database having NCBI General Identifier Nos. 3768668 and 701567.